Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo

To date, no gene transfer vector has produced prolonged gene expression fol lowing a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-repl icating Epstein-Barr virus (EBV)-based expression plasmids was developed. O ne plasmid contains the FR (EBV family of repeats) sequence and the express ed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lac ks FR. Although unable to replicate in mice, intravenous co-injection of EB V-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially p rolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vect or system could be intravenously re-injected multiple times into immunocomp etent mice without loss of transfection efficiency. Use of this vector syst em significantly improved the therapeutic efficacy of the biologically impo rtant human granulocyte colony-stimulating factor gene. Delivery of the hum an granulocyte colony-stimulating factor gene in EBV-based plasmids increas ed circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were ne ver elevated following injection of CLDCs lacking EBV-derived elements. Thu s, this EBV-based plasmid vector system both markedly prolongs gene express ion at therapeutic levels and efficiently and repeatedly re-transfects immu nocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus an d to block anti-EBNA-1 cytotoxic T cell responses.